Synthetic oligonucleotides complementary to mRNA or double-stranded DNA as a means to impair gene expression in living cells has provided the impetus to design and develop oligonucleotide analogues for therapeutic purposes. Unlike natural oligonucleotides, ,-oligodeoxyribonucleotides having alternative (3' 3')-and (5' 5')-internucleotidic phosphodiester linkages are not readily recognized by nucleases and, as a consequence of this inherent nucleolytic stability, may find application in antisense experiments. A simplified chemistry has been developed for the synthesis of -nucleoside precursors. The solid-phase synthesis of an ,- oligodeoxyribonucleotide having alternating (3'3')-and (5'5')- internucleotidic phosphodiester linkages (24-mer), complementary to a region overlapping the splice acceptor site of the second exon encoding the HIV-1 Tat gene product, has been accomplished. This oligomer hybridized to its complementary unmodified DNA strand and formed a complex having a Tm (53C) comparable to that obtained with a similar hybrid composed of the corresponding -oligonucleoside phosphorothioate and its complementary unmodified DNA strand (Tm=56C) but lower than that observed with the native DNA duplex (Tm=66C) under the same conditions. Thus, ,-oligodeoxyribonucleotides should exhibit similar sequence- specificity as the well-studied -oligodeoxyribonucleoside phosphorothioates. To provide better resistance against nucleases, ,- oligodeoxyribonucleotides having exclusively phosphorothioate linkages or only two these linkages at each terminus have also been synthesized. The potency and efficacy of these oligonucleotide analogues at inhibiting the replication of HIVIIIb in a human T-cell line are under investigation. Cytotoxic effects on the cell line are simultaneously assessed.